Brucella activates the host RIDD pathway to subvert BLOS1-directed immune defense

The phagocytosis and destruction of pathogens in lysosomes constitute central elements of innate immune defense. Here, we show that Brucella, the causative agent of brucellosis, the most prevalent bacterial zoonosis globally, subverts this immune defense pathway by activating regulated IRE1α-dependent decay (RIDD) of Bloc1s1 mRNA encoding BLOS1, a protein that promotes endosome–lysosome fusion. RIDD-deficient cells and mice harboring a RIDD-incompetent variant of IRE1α were resistant to infection. Inactivation of the Bloc1s1 gene impaired the ability to assemble BLOC-1-related complex (BORC), resulting in differential recruitment of BORC-related lysosome trafficking components, perinuclear trafficking of Brucella-containing vacuoles (BCVs), and enhanced susceptibility to infection. The RIDD-resistant Bloc1s1 variant maintains the integrity of BORC and a higher-level association of BORC-related components that promote centrifugal lysosome trafficking, resulting in enhanced BCV peripheral trafficking and lysosomal destruction, and resistance to infection. These findings demonstrate that host RIDD activity on BLOS1 regulates Brucella intracellular parasitism by disrupting BORC-directed lysosomal trafficking. Notably, coronavirus murine hepatitis virus also subverted the RIDD–BLOS1 axis to promote intracellular replication. Our work establishes BLOS1 as a novel immune defense factor whose activity is hijacked by diverse pathogens.


Introduction
Brucella is an intracellular vacuolar pathogen that invades many cell and tissue types, including nonprofessional and professional phagocytes (de Figueiredo et al., 2015). Brucellosis has eluded systematic attempts at eradication for more than a century (Godfroid et al., 2002), and even in most developed countries, no approved human vaccine is available (Ficht and Adams, 2009). The intracellular lifestyle limits exposure to host innate and adaptive immune responses and sequesters the organism from the effects of some antibiotics. Brucella evades intracellular destruction by limiting interactions of the Brucella-containing vacuole (BCV) with the lysosomal compartment (Criscitiello et al., 2013;Pizarro-Cerdá et al., 1998). BCVs harboring internalized Brucella traffic from endocytic compartments (eBCVs) to a replicative niche within vacuoles (rBCVs) that are decorated with markers of the endoplasmic reticulum (ER) (Pizarro-Cerdá et al., 1998;Starr et al., 2012). BCVs also accumulate autophagic membranes (aBCVs), which constitute a distinctive aspect of the intracellular lifestyle of the pathogen (Pandey et al., 2018;Starr et al., 2012). The VirB type IV secretion system (T4SS) is a significant virulence factor that regulates Brucella intracellular trafficking (Marchesini et al., 2011;Paredes-Cervantes et al., 2011;Sá et al., 2012;Smith et al., 2012). Brucella effectors secreted by the T4SS promote bacterial intracellular trafficking and growth via modulation of host functions (de Barsy et al., 2011;de Jong et al., 2008;Döhmer et al., 2014;Miller et al., 2017;Myeni et al., 2013) and organisms that lack this system fail to establish productive infections.
The Unfolded Protein Response (UPR) is an evolutionarily conserved signaling pathway that allows the ER to recover from the accumulation of misfolded proteins (Gardner et al., 2013;Walter and Ron, 2011) during ER stress. The UPR signals through the stress sensors IRE1α, ATF6, and PERK located in the ER membrane. When the luminal domains of these proteins sense unfolded proteins, they transduce signals to their cytoplasmic domains, which initiate signaling that ultimately results in UPR (Lee et al., 2008). IRE1α plays a central role in triggering UPR through an endonuclease/RNase activity in its cytoplasmic tail that catalyzes the splicing of Xbp1 mRNA, which is then translated to generate the XBP1 transcription factor (Lee et al., 2008;Ron and Walter, 2007). IRE1α RNase activity can also cleave a wide variety of cellular mRNAs that leads to their degradation in a process termed regulated IRE1-dependent mRNA decay (RIDD) (Hollien and Weissman, 2006). The RIDD pathway displays selectivity. For example, the pathway cleaves a specific subset of mRNAs encoding polypeptides destined for cotranslational translocation into the ER lumen. The degradation of these mRNAs supports ER homeostasis by reducing the flux of nonessential polypeptides into the ER (Hollien and Weissman, 2006). The molecular targets of RIDD activity, and the physiological roles that this process plays in cells, remain areas of investigation.
Brucella infection induces host cell ER stress and activates host UPR (de Jong et al., 2013;Pandey et al., 2018;Smith et al., 2013;Taguchi et al., 2015;Wang et al., 2016). The UPR sensor IRE1α, but neither PERK nor ATF6, is required for the intracellular replication of the pathogen (Qin et al., 2008;Taguchi et al., 2015), indicating that the IRE1α signaling pathway confers susceptibility to host cell parasitism. An IRE1α-ULK1 signaling axis also contributes to conferring susceptibility to Brucella intracellular replication; IRE1α-directed activation of components of the host autophagy program promotes proper bacterial intracellular trafficking and replication (Pandey et al., 2018). Despite the abovementioned advances, our understanding of how the IRE1α-RIDD axis and downstream processes regulate the intracellular lifestyle of Brucella remains largely unknown.
BLOS1 [biogenesis of lysosome-related organelles complex-1 (BLOC-1) subunit 1, also known as BLOC1S1/GCN5L1], a subunit of both the BLOC-1 and the BLOC-1-related complex (BORC), plays diverse roles in cells, including mitochondrial protein acetylation, modulation of metabolic pathways, and endosome-lysosome trafficking and fusion (Bae et al., 2019;Guardia et al., 2016;Pu et al., 2017;Pu et al., 2015). In mammalian cells, BLOS1 has also been shown to be a principal target of RIDD activity (Bae et al., 2019;Bright et al., 2015;Hollien et al., 2009) and is required for host cell cytotoxicity induced by Ebola virus (Carette et al., 2011). To date, the roles and mechanisms by which BLOS1 controls infection by intracellular pathogens remain largely unknown. Here, we demonstrate that IRE1α-directed Bloc1s1 mRNA degradation confers susceptibility to Brucella infection. Brucella-induced RIDD activity suppresses Bloc1s1 expression, disassembles BORC components, and limits BLOS1-regulated interactions between BCVs and lysosomes. In addition, we show that murine hepatitis virus (MHV), a betacoronavirus, also subverts BLOS1 activity to promote its intracellular replication. Collectively, these activities promote the productive subcellular trafficking and intracellular replication of diverse pathogens. Our findings, therefore, identify BLOS1 as a novel immune defense factor that defends against bacterial and viral infection and show that Brucella and MHV subvert this innate immune defense system to promote disease.

Ern1 conditional knockout mice are resistant to Brucella infection
Brucella induces host cell UPR during infection (Qin et al., 2008;Taguchi et al., 2015) and activates an IRE1α-to-autophagy signaling axis in host cells to promote its intracellular lifestyle (Pandey et al., 2018). To extend these findings to an in vivo model of brucellosis, we tested the hypothesis that UPR and IRE1α confer susceptibility to Brucella infection in mice harboring a conditional mutation in Ern1, the gene encoding IRE1α. Because mice homozygous for null mutations in Ern1 display embryonic lethality during organogenesis, we used a control Lyz2-Ern1 wt/wt [henceforth, wt (wild-type)-IRE1α] and a Ern1 conditional knockout (CKO mouse line (Ern1 flox/flox ; Lyz2 Cre/+ , hence after, m-IRE1α)) (Figure 1figure supplement 1A) (gift from the Iwawaki lab) in the experiments. In this line, exons 20-21 of the gene encoding IRE1α were deleted in monocytes and macrophages, generating animals in which the endonuclease domain (and hence RIDD activity) was specifically disrupted. However, the kinase domain remained intact Iwawaki et al., 2009). Macrophages are critical cellular targets for Brucella colonization (de Figueiredo et al., 2015). Hence, the tissue and molecular specificity of this lesion rendered the m-IRE1α mouse an ideal system for investigating how bacterial activation of host RIDD activity controls intracellular parasitism by the virulent B. melitensis strain 16M (Bm16M).
After confirming the RNase activity deficiency of the truncated IRE1α in bone marrow-derived macrophages (BMDMs) from m-IRE1α animals ( Figure 1-figure supplement 1B), we verified that m-IRE1α mice had normal organ morphologies, fertility, growth, and development. In addition, we showed that these animals had similar B cell (B220 + ), T cell (CD4 + or CD8 + ), and CD11b + profiles ( Figure 1-figure supplement 1C). We also showed that the IL-1β, IL-6, and TNF-α responses of BMDMs to LPS stimulation were reduced in mutant mice ( Figure 1A-C), consistent with previous findings that these LPS-mediated responses are controlled, in part, by IRE1α activity (Martinon et al., 2010).
To determine whether IRE1α activity in macrophages contributed to pathogen burden, dissemination, and disease progression, we infected wt-IRE1α control and m-IRE1α mice with Bm16M via the intraperitoneal route, humanely sacrificed the mice at various times postinfection, and then determined the bacterial burden in assorted tissues by quantifying the number of recovered colonyforming units (CFUs). We found that tissue-specific mutation of IRE1α resulted in enhanced resistance to bacterial infection with significant reductions in bacterial load in the spleen and liver compared to wt-IRE1α controls at 7 or 14 days postinfection (dpi), respectively ( Figure 1D, E). However, both infected wt-and m-IRE1α mice displayed similar spleen weights (Figure 1-figure supplement 1D, E) and spleen or liver inflammation ( Figure 1F-H), revealing that the lower numbers of CFU recovered from m-IRE1α animals were not accompanied by corresponding decreases in inflammation. To test the hypothesis that the differential bacterial burden in macrophage cells, the predominant cell type in which the pathogen resides and replicates in vivo, accounted for this reduction, we compared the bacterial load in CD11b + cells from control and m-IRE1α mice that had been infected with Bm16M for 7 days. We found that indeed CD11b + cells from the spleens of m-IRE1α mice displayed striking Figure 1. Host IRE1α is required for Brucella infection in vivo. Innate cytokine production of IL-1β (A), IL-6 (B), and TNF-α (C) in bone marrow-derived macrophages (BMDMs) from the wild-type (WT, wt-IRE1α) control and Ern1 conditional knockout (CKO, m-IRE1α) mice. The BMDMs were stimulated with LPS (100 ng/ml) and at 6 hr poststimulation the cytokine production of the treated cells was determined. Colony-forming unit (CFU) assay for B. melitensis 16M (Bm16M) intracellular survival in spleens and livers of wt-and m-IRE1α mice at 7 (D) or 14 (E) days post infection (dpi). (F) Histopathology of representative hematoxylin and eosin (H&E) stained sections of spleen and liver from Bm16M-infected wt-and m-IRE1α mice at 14 dpi. Bar: 100 μm.  Figure 1I), thereby suggesting that the resistance of these cells to intracellular parasitism contributed to the resistance phenotype observed at the organismal level. Divergent Brucella species display distinct host preferences; however, their interactions with host cells share common features (de Figueiredo et al., 2015). To test the hypothesis that m-IRE1α mice also displayed resistance to infection by other Brucella species, we infected these mice with B. abortus strain S2308 (BaS2308), a strain that displays tropism for cattle. We then assessed tissue burden in spleen and liver at 7 dpi. We found that m-IRE1α mice also exhibited resistance to BaS2308 infection ( Figure 1J), thereby indicating that the resistance phenotype of the mutant mice was not pathogen species specific.

IRE1α RNase activity confers susceptibility to Brucella infection
Xbp1 splicing was dramatically diminished in BMDMs from m-IRE1α mice (Figure 1-figure supplement 1B), indicating that BMDMs from the m-IRE1α mice carried the expected functional defects in IRE1α RNase activity. We thus tested the hypothesis that IRE1α RNase activity confers susceptibility to intracellular parasitism by Bm16M. First, we performed CFU assays of Bm16M infection of BMDMs from m-IRE1α and control mice and found that the replication efficiency of Bm16M in m-IRE1α BMDMs at 8, 24, and 48 h.p.i. was significantly lower than controls ( Figure 1K). Similar results were observed in control and Ern1 −/− mouse embryonic fibroblasts (MEFs) (Qin et al., 2008;Figure 1-figure supplement 2A-B). Second, we observed fewer Bm16M were recovered from BMDMs or RAW264.7 macrophages treated with 4μ8C, a compound that specifically antagonizes IRE1α RNase activity while leaving its kinase activity intact (Cross et al., 2012), than mock-treated controls ( Figure 1L-M). These findings supported the hypothesis that IRE1α RNase activity confers susceptibility to infection by regulating the intracellular trafficking and/or survival of the pathogen.

IRE1α regulation of Bm16M intracellular replication is XBP1 independent
We performed additional experiments to interrogate the role of IRE1α RNase activity in controlling Brucella infection. Two possibilities were explored: (1) IRE1α catalyzed splicing of Xbp1 transcripts, and downstream expression of XBP1-responsive genes, conferred susceptibility to Bm16M infection; or (2) IRE1α catalyzed RIDD activity controlled this process. To determine whether Xbp1 splicing was the reason, we examined the survival and intracellular replication of the pathogen in BMDMs harvested from Xbp1 −/− mice (gift from the Glimcher lab) in which Xbp1 was conditionally ablated from monocytes, macrophages, and granulocytes (henceforth, ∆Xbp1 mice) and found that Bm16M replicated similarly in ∆Xbp1 BMDMs and wt-Xbp1 littermate controls ( Figure 1N). Moreover, Bm16M displayed similar levels of liver and spleen colonization in ∆Xbp1 and wt-Xbp1 mice ( Figure 1O, P). These data suggested an XBP1-independent role for IRE1α RNase activity in conferring susceptibility to Bm16M infection, thereby implicating RIDD as the sought-after activity (see below).
Quantification of inflammation of spleens (G) or livers (H) at 14 dpi. (I) CFU assays of CD11b + cells from Bm16M-infected wt-or m-IRE1α mice. (J) CFU assay for B. abortus S2308 (BaS2308) intracellular survival in spleens and livers in wt-IRE1α control or m-IRE1α mice at 7 dpi. (K) Bm16M invasion and intracellular replication in BMDMs from m-IRE1α and control mice. h.p.i.: hours post infection. CFU assays of Bm16M infection of WT BMDMs (L) or RAW264.7 macrophages (M). Host cells were pretreated with 4μ8C (50 μM) 1 hr before and during infection; CFUs of the infected cells were determined at the indicated h.p.i. (N) CFU assays for Bm16M infection of BMDMs from WT and Xbp1 knockout (ΔXbp1) mice at the indicated h.p.i. CFU assay for Bm16M intracellular survival in spleen (O) or liver (P) in WT or ΔXbp1 mice at 14 dpi. Immunoblotting assay for IRE1α expression (Q) and quantification of the expression levels (R) in BMDMs during a time course (48 hr) of Bm16M infection. Bm16M infection induces phosphorylation of host IRE1α (S) and quantification of the phosphorylated levels of IRE1α during a time course (24 hr) of infection (T). Images/blots are representative of three independent experiments. Statistical data represent the mean ± standard error of mean (SEM) from three independent experiments. *, **, and *** indicate significance at p < 0.05, 0.01, and 0.001, respectively.
The online version of this article includes the following figure supplement(s) for figure 1:   To dissect the mechanism by which IRE1α activity confers susceptibility to intracellular parasitism by Bm16M, we examined this process in BMDMs derived from m-IRE1α mice. We observed that the expression level of IRE1α was relatively unchanged during a time course ( Figure 1S,T). Host IRE1α activity was also required for Brucella intracellular parasitism ( Figure 1K; Figure 1-figure supplement 2). To test whether IRE1α regulates Bm16M intracellular trafficking, we used confocal immunofluorescence microscopy (CIM) to analyze the localization of the pathogen in Ern1 +/+ and Ern1 −/− MEFs, or m-IRE1α and control BMDMs. In IRE1α harboring controls, the pathogen transiently trafficked through early and late endosomes (EEA1 + and M6PR + compartments, respectively) ( Our data, therefore, demonstrated that IREα activity controls Bm16M intracellular replication, likely via regulation of BCV ER trafficking. These findings encouraged us to investigate the molecular mechanisms driving these phenomena.

Brucella infection downregulates RIDD genes
We were intrigued with the hypothesis that Bm16M subverts host RIDD activity to promote intracellular parasitism. First, since host UPR/IRE1α RNase activity is induced by Brucella effectors secreted by the T4SS of the pathogen (de Jong et al., 2013), we tested whether RIDD activity was dependent upon the Brucella T4SS. We found that induction of IRE1α RNase activity occurred in a Brucella T4SSdependent fashion (Figure 3-figure supplement 1A). Next, we performed RNA-seq analysis to define candidate host genes whose transcripts were subject to RIDD control during Brucella infection. Specifically, we used Bm16M to infect triplicate sets of BMDMs as follows: (1) solvent control-treated wt-IRE1α, (2) 4μ8C-treated wt-IRE1α, or (3) solvent control-treated m-IRE1α ( Figure 3-figure supplement 1B). At 4 or 24 h.p.i., we harvested host mRNA for RNA-seq analysis. Differential expression analysis was then performed to identify genes that were downregulated following Bm16M infection of wt-IRE1α cells but were unchanged or upregulated in either infected, drug-treated cells, or infected, m-IRE1α cells. Genes that displayed reduced expression (p < 0.05) in response to infection at 4 and/or 24 h.p.i., and also whose infection-dependent reductions in expression were reversed upon treatment with 4μ8C, or in m-IRE1α cells, were defined as candidate 'RIDD genes'. This analysis resolved 847 candidate RIDD genes ( Figure  We performed several experiments or analyses to validate candidate RIDD genes identified in the RNA-seq analysis. First, we compared our list of candidate RIDD genes to genes previously reported to be subject to RIDD control (Bright et al., 2015;Han et al., 2009;Hollien et al., 2009;So et al., 2012). This comparison identified 40 genes that were previously shown to be substrates of IRE1α RNase activity and/or displayed expression patterns consistent with RIDD targeting ( Figure 3A, B). Second, we used real-time quantitative reverse transcription-PCR (qRT-PCR) to measure the expression levels of several candidate RIDD genes, including Bloc1s1, Cd300lf, Diras2, and Txnip ( Figure 3D-G; Figure 3-figure supplement 3) during infection. We found that the expression of these genes was significantly lower in Brucella-infected wt-IRE1α cells than in m-IRE1α cells ( Figure 3D-G). Third, we examined whether similar reductions in expression of candidate RIDD genes were observed in host cells infected with B. abortus S19 (BaS19, a vaccine strain) or BaS2308. We found that BaS19 or BaS2308 induced similar phenotypes as Bm16M (Figure 3-figure supplement 3A-D), suggesting that the phenotype was not species specific. To determine whether RIDD-mediated Txnip expression   was also regulated by the microRNA miR17, a molecule that affects TXNIP mRNA stability (Lerner et al., 2012), we measured the expression of miR-17 via qRT-PCR. We found that the expression of miR-17 was reduced (Figure 3-figure supplement 3E). Fourth, we used qRT-PCR to test the hypothesis that heat-killed bacteria induced similar changes in RIDD gene expression. We found that heat-killed bacteria did not cause a similar effect. Hence, the induction of RIDD activity in host cells required interactions with the viable agent ( Figure 3-figure supplement 3F) and was also T4SS dependent ( Figure 3-figure supplement 1A). Finally, we found that the expression of the key RIDD gene Bloc1s1 was also reduced in ∆Xbp1 BMDMs infected with Bm16M, or when ER stress was induced in these cells ( Figure 3H). These data suggested that the observed changes in host gene expression patterns were not a consequence of alterations in XBP1 transcription factor activity. Taken together, these data supported the hypotheses that (1) Bm16M infection induces RIDD activity in host cells and (2) RIDD activity confers enhanced susceptibility to intracellular parasitism by Brucella. However, these findings left open the question of the molecular mechanism by which RIDD activity controlled Bm16M replication.

RIDD activity on Bloc1s1 controls Brucella intracellular parasitism
Our RNA-seq analysis identified Bloc1s1 as a Brucella-induced RIDD gene. BLOS1 is a subunit of both the BLOC-1 and BORC complexes and plays diverse roles in cell physiological and biological processes, including endosome-lysosome trafficking and fusion ( Figure 3C; Bae et al., 2019;Guardia et al., 2016;Pu et al., 2017;Pu et al., 2015). However, the mechanisms by which Bloc1s1 regulates microbial infection are largely unknown. This fact encouraged us to test the hypothesis that Bloc1s1 plays a central role in regulating Bm16M intracellular parasitism. First, we generated a cell line carrying a nonfunctional Bloc1s1 mutant allele (mBloc1s1). Mammalian BLOS1 contains three conserved XAT hexapeptide-repeat motifs that are essential for acetyltransferase activity and may also be a necessary structure-defining feature for acetyl-CoA contact Wu et al., 2021a). Using CRISPR/cas9-mediated gene editing, we mutated the first XAT hexapeptide-repeat motif, which in the wild-type encodes 'EALDVH', and in the mutant encodes 'EVVDH or EVDH' (Figure 4-figure supplement 1A, Supplementary file 1). A cell line containing gene encoding Cas9 and a nonspecific gRNA was used as a control of the mBloc1s1 mutant line. Second, we generated a RIDD-resistant Bloc1s1 cell line (henceforth Rr-Bloc1s1). In this line, a mutation (from 'G' to 'U') was introduced into Bloc1s1 mRNA stem-loop structure (i.e., the target of IRE1α RNase activity) that rendered the mutated Bloc1s1 mRNA stem-loop structure-resistant cleavage by IRE1α RNase. A Bloc1s1::Bloc1s1-HA line that overexpresses Bloc1s1 (wt-Bloc1s1) served as a control of the Rr-Bloc1s1 cell line ( We characterized the developed cell lines in several ways. First, we noted that α-tubulin acetylation levels had been reported to be controlled, in part, by BLOS1 activity levels . Therefore, we monitored α-tubulin acetylation to assess whether our developed cell lines did, in fact, display alterations in BLOS1 activity. We found that mBloc1s1 and Rr-Bloc1s1 cells displayed reduced levels ( Figure 4A) and maintained relatively higher levels ( Figure 4B), respectively, of acetylated α-tubulin, compared to their corresponding controls. These data supported the hypothesis that these upper-left-corner panel: enriched KEGG pathways and interacting candidate RIDD genes (%). qRT-PCR validation of RIDD candidate genes Bloc1s1 (D), Diras2 (E), Cd300lf (F), and Txnip (G) identified from RNA-seq analysis. Relative mRNA expression levels in potential RIDD targets from control and m-IRE1α BMDMs infected with Bm16M at 16 and 48 h.p.i. were measured by qRT-PCR. (H) qRT-PCR analysis of expression levels of Bloc1s1 in ΔXbp1 BMDMs that were either uninfected (control), infected with Bm16M, or treated with tunicamycin (Tm, an UPR inducer, 5 μg/ml) at 4-hr post infection/ treatment. Expression levels of the indicated genes were normalized to Gapdh expression. Statistical data represent the mean ± standard error of mean (SEM) from three independent experiments. *, **, and ***: significance at p < 0.05, 0.01, and 0.001, respectively.
The online version of this article includes the following source data and figure supplement(s) for figure 3: Source data 1. Candidate RIDD gene identified in host cells infected by Brucella melitensis Bm16M.      . Finally, we monitored the expression levels of BLOS1 protein during a 48-hr time course of infection. We found that BLOS1 expression was reduced at 16 h.p.i., and continuously decreased during Bm16M infection in wt-IRE1α control cells; however, in m-IRE1α BMDMs, BLOS1 expression was relatively stable or increased (at 48 h.p.i.) ( Figure 4E). Similar results were observed in 4μ8C treated or untreated mBloc1s1, Rr-Bloc1s1, and control cells infected with BaS2308 ( Figure 4-figure supplement 1D, E). These data demonstrate that low or high BLOS1 expression levels promote or impair Brucella infection, respectively.

BLOS1 regulates Brucella intracellular trafficking
The mechanism by which BLOS1 regulates Brucella infection was unknown. However, the observed subcellular trafficking defect of the pathogen in host cells harboring mutant or deficient variants of IREα ( Figure 2; Figure 2-figure supplement 1) suggested that BLOS1 may control the intracellular parasitism of the pathogen by regulating its subcellular trafficking. To illuminate this aspect, we first characterized the mBloc1s1, Rr-Bloc1s1, and the corresponding control cell lines by treating them with tunicamycin (Tm, an UPR inducer) or 4μ8C, or infected them with Bm16M. We then assessed the trafficking of the pathogen in these cells using CIM. We found that low levels of BLOS1 or nonfunctional BLOS1 in uninfected or infected cells were associated with the accumulation of late endosome/ lysosome (LE/Lys) membranes in the vicinity of nuclei, reduced colocalization of latex beads with cathepsin D, and increased perinuclear LC3b index or autophagic activity near nuclei, in both control and Tm-treated conditions (Figure 4-figure supplement 2A, C, E, G). In these studies, the LC3b index was defined as: (Total number of identified cells with the ratio of the mean LC3b intensity in the cytoplasm to that in the nucleus <1)/(Total number of the analyzed cells). In contrast, overexpression of BLOS1 (the wild-type cells expressing wt-Bloc1s1 or Rr-Bloc1s1) reduced LE/Lys perinuclear accumulation, increased the localization of latex beads in cathepsin D + compartments, and reduced perinuclear autophagic activity (Figure 4-figure supplement 2B, D, F, H). These data indicated that cells deficient in BLOS1 or with RIDD resistance differentially control lysosome intracellular trafficking and autophagic activity. Although significant inhibition of BCV trafficking to lysosomes in the mBloc1s1 cells was not observed at 24 and 48 h.p.i., the mBloc1s1 cells supported enhanced BCV trafficking to ER compartments during bacterial infection, compared to that in the wild-type control cells, or 4μ8Ctreated mBloc1s1 cells ( Figure 4F-H). In contrast, Rr-Bloc1s1 cells displayed reduced BCV trafficking to ER compartments, but instead promoted BCVs trafficking to lysosomes during Bm16M infection, compared to controls ( Figure 4I-K).
Bloc1s1 variant (Rr-Bloc1s1) treated or untreated with 4μ8C (50 μM), Tm (5 μg/ml), or both for 4 hr. CFU assays for Bm16M infection of RAW264.7 cells in which Bloc1s1 is nonfunctional (C), RIDD resistant (D) at the indicated h.p.i. (E) BLOS1 degradation assay during Brucella infection (upper panel) and quantification of the relative BLOS1 expression level (compared to the level of the loading control GAPDH) (lower panel) at the indicated h.p.i. ns: no significance. Colocalization of BCV with calreticulin (CRC) or cathepsin D (CTD) (F) and quantification of BCV-CRC + (G) or BCV-CTD + (H) in control and mBloc1s1 cells treated with or without 4μ8C (50 μM) at the indicated h.p.i. Red asterisks: significance (p < 0.001) compared to control cells at 6 h.p.i. Colocalization of BCV with CRC or CTD (I) and quantification of BCV-CRC + (J) or BCV-CTD + (K) in the wt-Bloc1s1 and Rr-Bloc1s1 cells treated with or without 4μ8C (50 μM) at the indicated h.p.i. (L) Quantification of BLOS1 fluorescence integrated density (FID) per BCV in the mBloc1s1, Rr-Bloc1s1, or their corresponding control cells treated with or without 4μ8C (50 μM) at the indicated h.p.i. S: significance (p < 0.01) compared to that without 4μ8C treatment. Host cells were infected with or without Bm16M, and at the indicated h.p.i., the cells were harvested for immunoblotting assays or fixed and subjected to confocal immunofluorescence assays. Blots/images are representative of three independent experiments. Statistical data represent the mean ± standard error of mean (SEM) from three independent experiment. *p < 0.05; **p < 0.01; ***p < 0.001.
The online version of this article includes the following figure supplement(s) for figure 4:    To test the hypothesis that Bm16M infection alters the dynamics of associations between BLOS1 and BCVs, we used CIM approaches to localize these elements during a time course of infection after confirmation of the specificities of antibodies used in the work (see below) (Figure 4-figure supplement 3A-D). We found higher levels of BLOS1 colocalization with BCVs in 4μ8C-treated control or mBloc1s1 cells than their corresponding untreated cells ( Figure 4L); moreover, at 24 h.p.i., lower levels of BLOS1 + BCVs were observed in mBloc1s1 cells compared to wild-type controls (Figure 4figure supplement 3E, F). Rr-Bloc1s1 and/or 4μ8C inhibition of Bloc1s1 degradation in host cells (i.e., Rr-Bloc1s1, 4μ8C-treated wt-Bloc1s1 control or Rr-Bloc1s1 cells) significantly promoted BLOS1 + BCVs compared to the wt-Bloc1s1 control ( Figure 4L, Figure 4-figure supplement 3E, F). These findings demonstrated that nonfunctional BLOS1 permitted the trafficking of the pathogen from LE/ Lys membranes to the ER. However, Rr-Bloc1s1 cells (or 4μ8C-treated cells) promoted the trafficking and degradation of the pathogen in lysosomes.

Disassembly of BORC promotes BCV trafficking to and accumulation in the ER
To test whether BORC-related lysosome trafficking components mediate BCV trafficking during infection, we analyzed the dynamics of the interaction of BCVs with LAMTOR1 (a central component of mTORC1), the small GTPase ARL8b, and kinesin KIF1b and KIF5b proteins during infection. During bacterial intracellular trafficking and replication, colocalization of BCVs with both LAMP1 and LAMTOR1, LAMP1, or LAMTOR1 decreased in control and mBloc1s1 cells, whereas these interactions were observed at higher levels in cells harboring Rr-Bloc1s1 variants ( Figure 5A Similarly, recruitment of ARL8b to BCVs and/or LAMP1 was reduced in control and mBloc1s1 cells, which impaired their kinesin-dependent movement toward the cell periphery; however, these interactions were maintained in Rr-Bloc1s1 cells ( Figure 5C, D, F, H; Figure 5-figure supplement 1C-D). KIF1b + and KIF5b + preferentially drive lysosomes on peripheral tracks and perinuclear/ER tracks, respectively (Guardia et al., 2016). In control, mBloc1s1, and wt-Bloc1s1 cells, BCV interactions with KIF1b + or KIF5b + decreased ( Figure 6A, B BORC, a protein complex that contains three components (i.e., BLOS1, BLOS2, SNAPIN) shared with the BLOC-1 complex and five other proteins KXD1, C17orf59 (Lyspersin), LOH12CR1(Myrlysin), C10orf32 (Diaskedin), and MEF2BNB, plays a critical role in the regulation of lysosome positioning (Pu et al., 2015). In HeLa cells, interference with BORC triggers LE/Lys trafficking to the cell center via dynein, resulting in a characteristic clustering of LE/Lys in perinuclear regions (Pu et al., 2015). We hypothesized that degradation of Bloc1s1 mRNA by IRE1α during Brucella infection interferes with BORC assembly, resulting in the alteration of recruitment or disassociation of BORC-related trafficking components, and increased LAMP1 + -BCV perinuclear trafficking and fusion with the ER and/or macroautophagosome membranes. To test this hypothesis, we performed protein co-immunoprecipitation (Co-IP) assays to measure the association of BORC components with each other in Brucella-infected or -uninfected host cells. We found that in uninfected cells, BLOS1 interacted with protein components of BLOC-1 (PALLIDIN), BORC (KXD1), and both BLOC-1 and BORC (BLOS2, SNAPIN) ( Figure 7A, B). Moreover, under this condition, BORC components localized with peripheral or cytosolic LE/Lys membranes ( Figure 5-figure supplement 1; Figure 6-figure supplement 1). However, in Brucella-infected cells, where Bloc1s1 mRNA was degraded and BLOS1 protein depletion was observed ( Figure 3D, H; Figure 4G Figure 7C, D). In fact, interactions between LYSPERSIN and KXD1 in control cells were also only detected at early time points (2 h.p.i.), but not at later time points corresponding to intervals when bacterial intracellular trafficking and replication were expected to occur; these interactions were also hardly detected in infected cells expressing mBloc1s1 variants at these time points ( Figure 7C, E). The reduced interactions between BLOS1 and BORC component  Figure 7C-E). In Brucella-infected Bloc1s1 overexpressing (wt-Bloc1s1) cells, substantial reductions in the interactions were also observed ( Figure 7F, G). In the infected Rr-Bloc1s1 cells, the interaction was maintained at a relatively higher level ( Figure 7F, G), suggesting that the BORC complex remains assembled. The integrity or disassociation of BORC was consistent with the interactions between BORC-related trafficking components and with the colocalization dynamics of BCVs with BLOS1, mTORC1/LAMP1, LAMP1/ARL8b, LAMP1/KIF1b or with LAMP1/KIF5b. These findings were also consistent with BCV peripheral or perinuclear/ER trafficking and accumulation (Figures 5 and 6; Figure 4-figure supplement 3E, F). The results collectively suggested that the degradation of Bloc1s1 mRNA during pathogen infection resulted in the disassembly of BORC, which promoted BCV trafficking to and accumulation in the vicinities of nuclei and likely facilitated the fusion of BCVs with ER membranes in which bacteria replicated.

Host RIDD activity on BLOS1 promotes coronavirus intracellular replication
In light of the global COVID-19 pandemic, we tested whether RIDD-controlled BLOS1 activity is a target for subversion by coronaviruses. We infected control or host cells harboring alterations in this pathway with mouse hepatitis virus [MHV, a positive-strand RNA virus classified as a member of the Betacoronavirus genus (CoV)]. Notably, previous studies have shown that MHV infection induces host cell UPR and activates IRE1α RNase and Xbp1 splicing (Bechill et al., 2008), thereby suggesting the hypothesis that MHV infection of host cells activates RIDD activity. To test this hypothesis, mBloc1s1 or control host cells were untreated or treated with 4μ8C. Next, these cells were infected with MHV for 24 hr. Virus plaque-forming units (PFUs) and host Bloc1s1 expression were then measured. We found that viral PFUs were reduced in 4μ8C-treated cells. However, significantly increased PFU in mBloc1s1 cells at 24 h.p.i. compared to controls was observed ( Figure 7H). Expression levels of Bloc1s1 mRNA were dramatically reduced during infection ( Figure 7I). Collectively, these findings suggested that coronavirus MHV, like Brucella, subverts the host RIDD pathway to promote intracellular infection.

Discussion
RIDD, a fundamental component of UPR in eukaryotic cells, cleaves a cohort of mRNAs encoding polypeptides that influence ER stress, thereby supporting the maintenance of ER homeostasis. In this report, we found that Brucella infection subverts UPR, in general (Pandey et al., 2018;Qin et al., 2008;Smith et al., 2013;Taguchi et al., 2015), and RIDD activity on Bloc1s1, in particular, to promote intracellular parasitism. BLOS1, encoded by RIDD gene Bloc1s1, is a shared subunit of both BLOC-1 and BORC complexes (Pu et al., 2015). Mutation or a reduction in BLOS1 expression affects both BLOC-1 and BORC ( Figure 7A-H). The BLOC-1 complex is mainly involved in endosomal maturation and endosome-lysosome trafficking and fusion (John Peter et al., 2013;Pu et al., 2015;Scott et al., 2018). Our work does not rule out the possibility that the disassociation of BLOC-1 also affects Brucella intracellular parasitism, especially in the early stages of cellular infection.
Investigation of Brucella-mediated RIDD genes provides an avenue for understanding how the pathogen subverts or evades host functions to promote its intracellular lifestyle. For example, Brucella infection downregulates the expression of Txnip, a gene shown in this work to be subject to RIDD control and known to facilitate the intracellular survival of the pathogen (Hu et al., 2020). Cells undergoing ER stress following treatment with UPR inducers (e.g., thapsingargin, Tm) increase TXNIP protein levels and mRNA stability by reducing levels of the TXNIP destabilizing microRNA, miR-17 (Lerner et al., 2012). Therefore, RIDD might be expected to increase the stability of TXNIP mRNA by    attacking the miR-17 that affects TXNIP mRNA stability. However, a recent report demonstrated that in vitro cultivated host cells or mouse tissues infected by BaS2308 display reduced expression levels of TXNIP mRNA and/or protein as well as miR-17 (Hu et al., 2020). Consistent with these results, we found that both TXNIP mRNA and miR-17 were destabilized during Brucella infection. These findings suggest that the downregulation of TXNIP during Brucella infection is miR-17 independent. The functions of other RIDD genes and the mechanisms by which they regulate miRNAs and their targets during Brucella infection constitutes an interesting area for further investigation.
Our findings support a stepwise working model by which Brucella subverts the host RIDD pathway to facilitate intracellular parasitism by disrupting BORC-directed lysosomal trafficking ( Figure 7J).
First, Brucella enters host cell via the endocytic pathway ( Figure 7J, right portion). Bacterial infection also induces UPR in host cells, a process associated with activation of IRE1α kinase (Pandey et al., 2018;Taguchi et al., 2015) and RNase activities (Smith et al., 2013; this work) ( Figure 7J, step 1). Second, degradation of the RIDD target Bloc1s1 by IRE1α RNase activity results in depletion of BLOS1 proteins and reduced association with BORC components ( Figure 3D and Figure 4G, H; Figure 7A-H; Figure 7J, steps 2 and 3). Third, these events drive the trafficking of BCVs to the ER and their perinuclear accumulation ( Figure 7J, steps 4 and 5), mitigate further fusion of BCVs with cytosolic lysosomes, and limit BCV trafficking to LE/Lys in peripheral regions where these organelles possess enhanced degradative functions (Figures 5 and 6; Figure 4-figure supplement 3E, F; Figure 7J, dash-blue line). Finally, the accumulation of BCVs decorated with ER proteins increases due to the fusion of BCVs with ER membranes and/or with noncanonical macrophagosomes (Pandey et al., 2018;Starr et al., 2012;Taguchi et al., 2015; Figure 7J, steps 5 and 6). These final events support the intracellular replication of the pathogen ( Figure 7J).
Several lines of evidence support the proposed mechanism. First, Brucella infection activates IRE1α RNase activity as evidenced by Xbp1 mRNA splicing (Figure 3-figure supplement 1A). However, the intracellular replication of the pathogen is not impaired in Xbp1 KO cells and mice ( Figure 1N-P). These findings support the hypothesis that IRE1α RNase activity is required for Brucella infection in an IRE1α-XBP1 independent fashion. Second, in addition to splicing Xbp1, IRE1α cleaves other mRNAs, resulting in their RIDD-mediated decay (Bae et al., 2019). We identified several mRNAs, including Bloc1s1 ( Figure 3A-C), that contain predicted stem-loop structures that were inferred to be targets of IRE1α RNase activity (Moore and Hollien, 2015). The expression of these mRNAs was downregulated in response to Brucella infection. Host cells harboring nonfunctional Bloc1s1 mutants were highly susceptible to pathogen infection, whereas cells that express a RIDD-resistant Bloc1s1 variant were resistant to Brucella infection ( Figure 4E, F).
Third, lysosome positioning regulated by BORC is a critical determinant of its functions. BORC associates peripherally with lysosomal membranes, where it recruits the small GTPase ARL8b to lysosomes. BORC and ARL8b promote lysosome movement by coupling to kinesin-1 (KIF5b) or kinesin-3 (KIF1b), which preferentially moves lysosomes on perinuclear tracks enriched in acetylated α-tubulin or on peripheral tracks enriched in tyrosinated α-tubulin, respectively (Guardia et al., 2016;Pu et al., 2015). Interference with BORC or other components of this pathway drives lysosome trafficking to the cell center via dynein. Thus, cells lacking BORC display a perinuclear clustering of lysosomes (Pu et al., 2015). Ragulator (a GEF for the Rag GTPases that signal amino acid levels to mTORC1) directly interacts with and inhibits BORC functions (Pu et al., 2017). Building upon these observations, we show that Brucella infection results in Bloc1s1 degradation and disassembly of BORC; moreover, during Brucella intracellular trafficking and replication, colocalization of mTORC1, ARL8b, and KIF1b with BCVs or lysosomes was reduced in control cells, and in cells expressing nonfunctional variants of BLOS1; however, KIF5b localization with BCVs or lysosomes was increased or in a higher level in these indicated h.p.i. showing in A, B and C, D, respectively. Dynamics of LAMP1-KIF1b + (G) or LAMP1-KIF5b + (H) in a time course (48 hr) of Bm16M infection at the indicated h.p.i. Host cells were infected with or without Bm16M, and at the indicated h.p.i., the cells were fixed and subjected to confocal immunofluorescence assays. Images are representative of three independent experiments. Statistical data represent means ± standard error of mean (SEM) from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.
The online version of this article includes the following figure supplement(s) for figure 6:   Figure 6-figure supplement 1). Colocalization of the BORC-related lysosome trafficking factors (i.e., ARL8b, KIF1b, and mTORC1) with BCVs or lysosomes in cells expressing Rr-Bloc1s1 variants were maintained at a relatively higher level (Figures 5 and 6). These findings demonstrate that blocking BORC function via the disassembly of the BORC complex through depletion of Bloc1s1 by Brucella infection drives BCVs toward the perinuclear region and ER accumulation, which likely facilitates the fusion of BCVs with the ER, thereby supporting intracellular parasitism.
Finally, RIDD-mediated Bloc1s1 degradation may promote BCV fusion with autophagosomes. Nutrient-starved cells display perinuclear clustering of lysosomes, which influences autophagosome formation and autophagosome-lysosome fusion rates (Korolchuk et al., 2011). Lysosome perinuclear clustering during starvation, ER stress induced by accumulation of misfolded proteins, drug treatments, and pathogen infection can disrupt metabolic homeostasis, thereby necessitating the induction of cell biological processes that return the cell to equilibrium. Macroautophagy and degradation of sequestered cytosolic materials by fusion of autophagosomes/macrophagosomes with lysosomes can promote the re-establishment of homeostasis (Bae et al., 2019;Korolchuk et al., 2011;Pu et al., 2017). Degradation of Bloc1s1 mRNA by IRE1α leads to the perinuclear accumulation of LE/Lys in response to ER stress in mouse cells. Overriding Bloc1s1 degradation results in ER stress sensitivity and the aggregation of ubiquitinated proteins. The LE/Lys perinuclear-trafficking and LE-associated endocytic transport promote the efficient degradation of these protein aggregates. Therefore, Bloc1s1 regulation via RIDD facilitates LE-mediated autophagy of protein aggregates, thereby promoting cell survival during stress (Bae et al., 2019). Hepatocytes from Bloc1s1 liver-specific knockout (DelVecchio et al., 2002) cells accumulate autolysosomes and lysosomes. In LKO hepatocytes, the initiation or extension of lysosomal tubules is abolished, which impairs autophagic lysosome reformation and results in the accumulation of enlarged autolysosomes (Wu et al., 2021b). Bloc1s1 degradation by the RIDD pathway promotes BCV perinuclear or ER-region clustering and may also avoid the peripheral movement of BCVs away from the ER region as a consequence of reduced α-tubulin acetylation. These processes may facilitate BCV fusion with ER membranes or (macro)phagosomes, promote the enlargement of aBCVs and further bacterial replication, and ultimately relieve Brucella-induced ER stress (Pandey et al., 2018;Qin et al., 2008;Starr et al., 2012;Taguchi et al., 2015).
In addition to Brucella, the betacoronavirus MHV also subverts the RIDD-BLOS1 axis to promote intracellular replication ( Figure 7H, I), thereby indicating that RIDD control of BLOS1 activity is not pathogen specific. How the host RIDD-Bloc1s1 axis regulates interactions between host cells and coronaviruses merits further investigation. However, additional possibilities for regulatory control can be envisioned. First, coronaviruses utilize many proteins such as nsp1 to inhibit host protein synthesis in the first 6 hr of infection (Nakagawa and Makino, 2021). Second, BLOS1 contains a potential coronavirus 3C-like protease cleavage site, LQ^SAPS, near its C-terminus, thereby rendering it potentially susceptible to direct subversion by coronaviral pathogens. Finally, coronaviruses have evolved to subvert host interferon defenses (Thoms et al., 2020), which may contribute to immune evasion. Future work will be directed toward examining these possibilities and the roles and mechanisms by which the RIDD-Bloc1s1 axis controls these and other host-pathogen interactions.

Materials and methods
All the key resources used in the work are listed in Key resources table used in this work. indicated h.p.i., the infected or uninfected host cells were harvested for Co-IP and immunoblotting assays, or qRT-PCR assays. Blots are representative of three independent experiments. Statistical data represent the mean ± standard error of mean (SEM) from three independent experiment. *p < 0.05; **p < 0.01; ***p < 0.001. Red asterisks: Compared to the same Brucella-infected cells at 2 h.p.i.

Figure 7 continued
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Continued
Continued on next page (FCS). For BMDMs, the abovementioned DMEM with 20% L929 cell supernatant, 10% FBS, and antibiotics was used. Cells were seeded in 24-or 96-well plates and cultured overnight before infection. For antibiotic protection assays, 1.25 × 10 5 (BMDMs) or 2.5 × 10 5 (RAW264.7) host cells were seeded in each well; for fluorescence microscopy assays, 1 × 10 4 or 5 × 10 4 cells were seeded in 96-well plates or on 12 mm glass coverslips (Fisherbrand) placed on the bottom of 24-well microtiter plates, respectively; for host RNA analysis, 1 × 10 5 host cells were seeded in each well of 24-well plates before infection. Host cells were infected with Brucella at an MOI of 100, unless otherwise indicated. Infected cells were then centrifugated for 5 min (200 × g) and incubated at 37°C. Thirty minutes to 1 hr postinfection, culture media was removed, and the cells were rinsed with 1× phosphate buffered saline (PBS, pH 7.4). Fresh media supplemented with 50 μg/ml gentamicin was then added for 1 hr to kill extracellular bacteria. Infected cells were continuously incubated in the antibiotic. At the indicated time points post infection, viable bacteria in infected cells were analyzed using the antibiotic protection assay or the immunofluorescence microscopy assay as previously described (Pandey et al., 2018;Qin et al., 2008).  (Iwawaki et al., 2009). Western blot analysis using anti-IRE1α antibodies (Novus Biologicals) and Xbp1 splicing were performed on BMDMs from CKO and control mice to validate the absence of full-length IRE1α in CKO mice.

BMDM harvest and cultivation
BMDMs collected from the femurs of Ern1 CKO and control mice were cultivated in L929-cell conditioned media [DMEM medium containing 20% L929 cell supernatant, 10% (vol/vol) FBS, penicillin (100 U/ml) and streptomycin (100 U/ml)]. After 3 days of culture, nonadherent precursors were washed away, and the retained cells were propagated in fresh L929-cell conditioned media for another 4 days. BMDMs were split in 24-well plates (2.5 × 10 5 cells/well) in L929-cell conditioned media and cultured at 37°C with 5% CO 2 overnight before use.

Whole animal infections with Brucella and histologic analysis
Mice from CKO and littermate control groups (5 mice/group or treatment) were intraperitoneally infected with B. melitensis and B. abortus (Bm16M and BaS2038, respectively) with a dose of 1 × 10 6 CFU. At 7 and 14 dpi, infected mice were euthanized, and the bacterial burden was assessed in spleen and liver. A portion of the tissue was fixed, and paraffin embedded for histopathological examination following hematoxylin and eosin staining. Sections were evaluated and scored for lesion severity (inflammation) using the previously described scoring system (Lacey et al., 2018), that is, 0 = no inflammation; 1 = minimal with inflammation involving <5% of tissue; 2 = moderate with focally extensive areas of inflammation (5-25% of tissue and involving 1 or more tissues); 3 = moderate to severe with focally extensive areas of inflammation (>25% to 50% of tissue and involving multiple tissues); and 4 = severe with large confluent areas of inflammation (>50% of tissue and involving multiple tissues). To assess Bm16M tissue burden, spleen or liver tissues were homogenized and subjected to a serial dilution. Finally, the diluted tissue homogenates (200 μl) were plated on TSA solid plates and CFUs were determined at 48-to 60-hr postincubation at 37°C in 5% CO 2 .

Latex bead phagocytosis assays
Phagocytosis assays for testing the phagocytic uptake and route of a substrate in the nonfunctional and RIDD-resistant Bloc1s1 variants in RAW264.7 murine macrophages were performed using the Phagocytosis Assay Kit (IgG FITC) (Cayman Chemical, USA) according to the manufacturer's instructions.

Bioinformatic analysis
KEGG pathway enrichment analysis was performed by using WebGestalt-2013 (WEB-based Gene Set Analysis Toolkit) (Wang et al., 2013) with RIDD genes. Gene Ontology (GO) Enrichment was performed by feeding the significantly DEGs to PANTHER-v7 (Gaudet et al., 2011;Mi et al., 2010) in all three classes: Molecular Function, Biological Process, and Cellular Component. Both KEGG pathway and GO enrichment analysis were filtered and sorted by Fisher test and Benjamini and Hochberg adjustment. The GeneCards website (Stelzer et al., 2016) was used to connect to the REFSEQ mRNAs from NCBI's GenBank. The FASTA option was chosen and the fasta file was saved. The fasta file was uploaded to RNAfold Web Server (Institute for Theoretical Chemistry at University of Vienna) with the fold algorithm options of minimum free energy and partition function and avoid isolated base pairs. The Forna option was then used to view the secondary structure and manually searched for a stem loop with NNNNNCNGNNGNNNNNN. Interaction network analysis of KEGG pathways and the RIDD genes (p < 0.05) identified in this work was visualized using Cytoscape (https://cytoscape.org/) as described previously (Zhao et al., 2017).
Generation of nonfunctional and RIDD-resistant Bloc1s1 variants in RAW264.7 murine macrophages Nonfunctional Bloc1s1 variants in RAW264.7 murine macrophages were generated using a protocol previously described (Hoffpauir et al., 2020). One clone containing either an amino acid deletion substitution or deletion in one of the XAT regions of murine BLOS1 (Figure 4-figure supplement 1A) was selected. For generation of Rr-Bloc1s1 variant and its control (wt-Bloc1s1) in RAW264.7 murine macrophages, RNA was first extracted using RNeasy Plus mini-kit. Bloc1s1 cDNA was generated from mRNA and cloned into pCR 2.1-TOPO vector. Site-directed mutagenesis was utilized to generate a g449t mutation into one of the Bloc1s1 plasmid clones (Figure 4-figure supplement 1B). Both the wild-type (WT) and mutated Bloc1s1 segments were removed from the pCR 2.1-TOPO vector and cloned into pE2n vectors. Gateway cloning was used to generate expression vectors pLenti-CMV-Hygro-DEST (w117-1)-Blos1-WT and pLenti-CMV-Hygro-DEST (w117-1)-Blos1-G449T. At every step post cDNA creation, plasmid amplification vectors were verified via sequencing. The plasmids were then transfected into Lenti-X cells with psPAX2 and VSVG packaging plasmids. The virus was collected at 24-and 48-hr post-transfection and stored at −80°C. RAW264.7 cells were transduced with the virus and cells containing the wt-or Rr-Bloc1s1 expression cassette were selected using hygromycin B (500 μg/ml).

Drug treatments
Host cells were coincubated in 24-well plates with tunicamycin or 4μ8C at the indicated concentrations. Cells were treated with drugs 1 hr before, and during, infection with the indicated Brucella strains and incubated at 37°C with 5% CO 2 . At the indicated time points postinfection, the treated cells were fixed with 4% formaldehyde and stained for immunofluorescence analysis or lysed to perform CFU assay or for RNA extraction assays as described above. To investigate whether the drugs inhibit Brucella growth, the drugs were individually added to Brucella TSB cultures at 37°C and incubated for 1 and 72 hr. CFU plating was used to assess bacterial growth in the presence of drugs, and thereby to evaluate the potential inhibitory effects. Host cells in which drug treatment or Brucella infection induced no significant differences in viability and membrane permeability as well as drugs that have no adversary effect on Brucella growth were used in the experiments reported in this work.

CIM assays
Immunofluorescence microscopy staining and imaging methods to determine Brucella intracellular trafficking and colocalization of the bacteria and host BORC components in infected host cells were perform as previously described (Pandey et al., 2017;Pandey et al., 2018;Qin et al., 2011;Qin et al., 2008) with minor modifications. Briefly, to visualize Bm16M intracellular trafficking, the indicated host cells (5.0 × 10 4 for 24-well plates and 1 × 10 4 for 96-well plates) were seeded on 12 mm coverslips placed on the bottom of wells of 24-or 96-well plates (without coverslip) and infected with Bm16M-GFP or Bm16M. At 0.5 (for 24-well plates) or 1 (for 96-well plates) h.p.i., the infected cells were washed with 1× PBS and fresh media containing 50 μg/ml gentamicin was added to kill extracellular bacteria. At the indicated time points post infection, the infected cells were fixed with 4% formaldehyde at 4°C for overnight or for 20 min at 37°C before confocal fluorescence or immunofluorescence microscopy analysis was performed as previously described (Pandey et al., 2017;Pandey et al., 2018;Qin et al., 2011;Qin et al., 2008). The primary antibodies used were listed in the Key Resources Table. Samples were stained with Alexa Fluor 488-conjugated, Alexa Fluor 594-conjugated, and/or Alexa Fluor 647 secondary antibody (Invitrogen/Molecular Probes, 1:1000). Acquisition of confocal images, and image processing and analyses were performed as previously described (Pandey et al., 2017;Pandey et al., 2018;Qin et al., 2011;Qin et al., 2008). The BioTek Cytation 5 and Gen 5 software (version 3.05) were used to calculate perinuclear Lamp1 index and autophagic flux. Specifically, for perinuclear Lamp1 index, the area of the nucleus and average intensity for Lamp1 was measured, noted as A N and Int N , respectively. The area of the whole cell and the average intensity for Lamp1 was measured, noted as A WC and Int WC , respectively. Next the area of the cell 3 µm off from the nucleus (peripheral region) and the average intensity for Lamp1 was measured, noted as A C and Int C , respectively. Then, Int WCN (average intensity of Lamp1 in the whole cell minus nucleus) and Int P (average intensity of Lamp1 in the perinuclear region) were calculated as the following formula: If Int P /Int WCN ≤ 1, which means that there is no or very little perinuclear colocalization; if Int P /Int WCN > 1, which means that there is perinuclear colocalization.
For autophagic activity, using BioTek Cytation 5 and Gen 5 software (version 3.05), the mean intensity of LC3b in the nucleus (M1) and in the cytoplasm (M2) were measured using a primary and secondary mask in every individual cell. In the Gen 5 software, a subpopulation analysis was carried out to identify cells that had a ratio of M2/M1 <1. From this, the perinuclear LC3b index or autophagic activity was calculated as the following formula: Perinuclear LC3b index (or autophagic activity) = (Total number of identified cells with M2/M1 <1)/Total number of the analyzed cells.
For calculation of the relative BLOS1 fluorescence integrated density (FID) per BCV, the corrected total cell fluorescence was measured by taking the integrated density (area of cells × mean fluorescence), then the number of Brucella in each cell and colocalization of BLOS1 and Bm16M (BCV-BLOS1 + , %) were counted. Cells that did not contain bacteria were removed from the calculation. The integrated density was divided by the number of bacteria in the cell to obtain the 'Total fluorescence per bacteria'. The 'relative BLOS1 FID per BCV' was then calculated by multiplying the 'Total fluorescence per bacteria' with the colocalization percentage. Since BLOS1 protein is more stable in control cells treated with 4μ8C, the value of the relative BLOS1 FID per BCV was normalized as 1.

Protein pull-down assays and immunoblotting analysis
Pull-down assays for testing physical interaction of proteins were perform using the Pierce Co-Immunoprecipitation (Co-IP) Kit (Thermo Scientific, USA) according to the manufacturer's instructions. Preparation of protein samples and immunoblotting blot analysis were performed as described previously (Ding et al., 2021;Pandey et al., 2017;Pandey et al., 2018;Qin et al., 2011). Densitometry of blots was performed using the ImageJ (http://rsbweb.nih.gov/ij/) software package. All Westerns were performed in triplicate and representative findings are shown.

Statistical analysis
All the quantitative data represent the mean ± standard error of mean from at least three biologically independent experiments, unless otherwise indicated. The data from controls were normalized as 1% or 100% to easily compare results from different independent experiments. The significance of the data was assessed using the Student's t-test (for two experimental groups), two-way analysis of variance test with Holm-Sidak's multiple comparisons, or the Kruskal-Wallis test with Dunn's multiple comparison. For the RNA-seq results, Log 2 fold changes were calculated and results were screened to meet the threshold (|log 2 FC (fold change)| > 1, p < 0.05) for selection. DEGs met the criteria and in UPRsome were included in the final lists.

Key resources
All key resources, including bacterial strains, mammalian cell lines, reagents, etc. used in this work are listed in Key resource table.

Additional files
Supplementary files • Supplementary file 1. Table S1. Recombinant plasmids constructed in this work.
• Supplementary file 2. Table S3. Primers used in this work.
• Transparent reporting form • Source data 1. Source Western blot and agrose gel images in PDF format.
• Source data 2. Original images of Western blots and agarose gels.

Data availability
All data generated or analyzed during this study are included in the manuscript, supporting file, and source data file.